We have shown that a variety of antimicrobial peptides (AMPs) and nuclear binding proteins that mimic chemokines also have the capacity to rapidly activate host immune responses. We have proposed calling these early warnings signals alarmins. Alarmins are characterized by having in vitro chemotactic or in vivo recruitment activity for cells expressing GiPCR, together with the capacity to interact with other receptors resulting in the activation of immature dendritic cells (iDC) into mature antigen-presenting capable of interacting with T lymphocytes. These alarmins, if administered together with an antigen result in considerable augmentation of both in vivo cellular and humoral immune responses to the antigen. We previously identified both alpha and beta types of defensins as alarmins with chemotactic and activating effects on immature dendritic cells (iDCs) as having in vivo immunoadjuvant effects. Some of the beta defensins interact with the CCR6 chemokine receptor, others with CCR2, while alpha defensins interact with an as yet unknown G-Protein Coupled Receptors (GiPCR). Defensins by binding to DNA also can activate DC's by triggering TLR-9. Indeed, human neutrophil peptide 1, HNP1 was recently reported by our collaborators to activate pDC's using NF-kB and IRF signaling pathways. The resultant IFN production presumably has considerable proinflammatory effects. Another antimicrobial peptide known as cathelicidin (LL37) and its murine homologue CRAMP are chemotactic for FPR2 receptors expressed on monocytes and precursors of iDC. Cathelicidins also induce the maturation iDC and are equally as potent adjuvants in vivo as alum. In addition, we have previously also identified eosinophil derived neurotoxin (EDN, a ribonuclease), granulysin from lymphocytes, lactoferrin from neutrophils and HMGB1, a nuclear binding protein, as functional alarmins. Although alarmins are structurally distinct, they are preformed and constitutively available. Alarmins are rapidly released from granules, cytosol or nucleus of leukocytes and epithelial cells or by necrotic cells. Alarmins can also be induced to be produced in response to proinflammatory stimulants by keratinocytes or epithelial cells lining the GI tract, GU tract and tracheobronchial tree. As such, alarmins probably represent an early warning system to alert the host defense to danger signals. During the past several years, we have also identified a transcription factor, High Mobility Group Nucleosome-binding protein-1 (HMGN-1), as an extracellular alarmin that is chemotactic for DC and an inducer of Toll-like receptor 4 (TLR-4) dependent immune responses. HMGN-1 has the capacity to recruit and induce the maturation of dendritic cells (DC) at sites of injection. HMGN-1 activates NF kappa B and multiple MAP kinases largely in a TLR4 dependent manner. Upon co-administration with antigens, HMGN-1 has potent adjuvant effects favoring Th1 immune responses. Conversely, mice genetically engineered to be deficient in HMGN-1 had greatly reduced antigen specific immune responses even in response to antigens administered together with LPS. This immune deficiency of HMGN-1 knockout mice was associated with deficient recruitment of inflammatory cells and DC to sites of immunization and reduced cytokine production by DC. Thus, HMGN-1 which is largely derived from non-leukocytes (e.g. epithelial cells) plays a non-redundant critical role in the development of innate and adaptive immune responses. In a previous experimental study, we showed that intraperitoneal administration of anthrax vaccine with HMGN1 protein markedly increased the production of both primary and secondary protective IgG antibodies to anthrax toxin. We previously showed that HMGN-1 knockout mice exhibit reduced resistance to tumor (EG-7 or EL-4) challenge. Conversely, tumor cells (EG-7 or EL-4) when transfected to overexpress HMGN1 showed a marked reduction in the rate of growth in normal mice. These observations indicated that HMGN1 is capable of augmenting tumor immunity. To maximize the adjuvant effects of HMGN1, we covalently linked it to a gp100 melanoma tumor antigen. Antigens fused to adjuvants have been shown to be delivered more effectively to the appropriate intracellular compartments of antigen presenting cells (APC's) resulting in improved antigen processing and presentation and greater T cell activation. We have immunized mice with gp100 linked to HMGN1 in the form of plasmid DNA using gene gun technology. This succeeded in inducing about 70% of the immunized mice to be resistant to a challenge with B16 melanoma tumor cells. However, therapy of mice with this plasmid DNA, which had been injected with B16 melanoma tumor cells four days previously, failed to inhibit tumor growth. We therefore injected a recombinant HMGN1 protein directly intratumorally into CT26 colon tumors in mice to proximate the adjuvant and antigen. This therapeutic vaccine trial did have a significant beneficial effect in slowing the tumor growth and prolonging the survival of mice, but did not cure any of the mice. We therefore have improved the delivery of the tumor vaccine and employ it in conjunction with other antitumor therapies to cure mice with larger tumors. We have been able to show that intratumoral injections of several TLR ligands together with checkpoint inhibitors cures mice with five types of tumors and results in their subsequent resistance to re-challenge with these tumors. We used R848, a TLR7/8 ligand, together with HMGN1, a TLR4 ligand, because they synergistically stimulated the maturation of dendritic cells and markedly increased their production of IL-12 and TNF. The combination of immunotherapeutic agents consisting of HMGN1, R848 (Resiquimod), a checkpoint inhibitor such as anti PDL-1 or anti CTLA4 antibody or a low dose of cytoxan successfully cured large (1cm diam.) tumors of the colon (CT26), kidney (RENCA), thymoma (EG7) lung (Lewis Lung) and liver (Hepa1-6) in mice. We have also developed a means of delivering the HMGN1 and R848 on gold nanoparticles intravenously with success in curing large colon and liver tumors. In collaboration with Professor Lin and her colleagues at the Traditional Chinese Medicine (TCM) Guang'anmen Hospital, Beijing, China we have tested a number of TCM's over the past decade in an effort to detect any with immunostimulant effects. We finally determined that a purified and characterized TCM known as Cryptotanshinone (CT) not only had cytotoxic effects on tumor cells, but also activated dendritic cells in vitro to mature and produce cytokines (e.g. IL-12 and TNF). We also were able to show that CT had the capacity to cure mice with large (1 cm diam.) colon tumors, when injected either intratumorally or intravenously together with a checkpoint inhibitor. In response to a request by Dr. Michael Zasloff, we showed that alpha-synuclein (alpha-S) was a potent chemotactic protein for neutrophils and monocytes. This result helped to understand the high expression of alpha-S within the human enteric nervous system in biopsy specimens from patients with a variety of inflammatory conditions of the intestinal wall characterized by infiltrations of acute neutrophil and chronic mononuclear inflammatory cells. Subsequently we also demonstrated that alpha-S was also a potent activator of dendritic cells and therefore functioned as an alarmin. This is of particular interest since mutations of alpha-S are associated with the development of familiar Parkinson's Disease.